One cause of patient morbidity and mortality, idiosyncratic drug toxicity remains a serious safety concern in both clinical drug development and after market launch. These idiosyncratic drug reactions can lead to restricted use and even withdrawal from the market, which consequently results in higher development cost for the pharmaceutical industry. For example, troglitazone, benoxaprofen and zomepirac were withdrawn from the market shortly after their release due to unacceptable toxicity profiles.
Idiosyncratic drug reactions are a rare event that usually shows in a high degree of individual susceptibility. In addition, these reactions are usually not dose-dependent. Currently, there are no animal models that can be used to evaluate such reactions that exclusively occur in humans. Therefore, idiosyncratic drug toxicities cannot be effectively evaluated in preclinical studies, and are often unnoticed in clinical trials.
At present, the mechanisms of idiosyncratic drug reactions are not well understood. There is a substantial amount of evidence to suggest that chemically reactive metabolites are involved in idiosyncratic toxicities, especially for liver toxicity. All drugs associated with idiosyncratic toxicity form reactive metabolites via various metabolic pathways mediated predominately by cytochrome P450 enzymes (CYPs), as well as by other oxidative enzymes such as peroxidases, cyclooxygenases and myeloperoxidases. It is hypothesized that drugs associated with such toxicities first undergo metabolic activation to generate toxic reactive metabolites that covalently bind to cellular proteins. These covalently modified proteins are immunogenic and thus trigger an immune response, resulting in idiosyncratic drug reactions. An alternative hypothesis states that covalent modifications of cellular proteins by reactive metabolites impair signal transduction cascades and vital functions of cells, leading to severe consequences observed in clinic. Thus there remains a need for methods for identifying reactive metabolites.
Chemically reactive metabolites can be classified into two categories based on their chemical properties—“soft” and “hard” reactive metabolites. “Soft” reactive metabolites comprise a majority of electrophilic metabolites which include quinones, quinone imines, iminoquinone methides, epoxides, arene oxides and nitrenium ions, and readily react with “soft” electrophiles such as the sulfhydryl group in cysteine. In contrast, “hard” reactive metabolites, most commonly seen as aldehydes, preferentially react to “hard” electrophiles such as amines of lysine, arginine and nucleic acids. Because of their instability, direct detection and characterization of reactive metabolites has proven to be extremely difficult. A commonly utilized approach is to trap reactive metabolites with a capture molecule, resulting in formation of a stable adduct that can be subsequently characterized by known detection methods, for example by tandem mass spectrometry.
Recently, Avery, Michael, J., in European Patent Publication EP 1,150,120 disclosed a high-throughput screening method for identifying test compounds producing reactive metabolites. The method comprises incubating a test compound with a microsomal drug metabolizing enzyme system in the presence of glutathione and detecting glutathione adducts formed therefrom using tandem mass spectrometry.
This method however, will identify reactive metabolites as well as non-reactive components, (including both unreactive metabolites and components of the reaction mixture), formed as a result of common response in mass spectroscopy detection, thus resulting in false positives.
Yan et al., in US Patent Publication 2005 0287623 A1 disclose a method for detecting reactive metabolites using stable isotope trapping and mass spectroscopy. However, the method as disclosed in Yan et al., detects only “soft” metabolites, but does not simultaneously detect both “hard” and “soft” reactive metabolites.